U.S. patent application number 11/284343 was filed with the patent office on 2006-07-27 for method for ligating nucleic acids and molecular cloning.
This patent application is currently assigned to Stratagene California. Invention is credited to Carsten-Peter Carstens, Joseph A. Sorge.
Application Number | 20060166332 11/284343 |
Document ID | / |
Family ID | 46323217 |
Filed Date | 2006-07-27 |
United States Patent
Application |
20060166332 |
Kind Code |
A1 |
Sorge; Joseph A. ; et
al. |
July 27, 2006 |
Method for ligating nucleic acids and molecular cloning
Abstract
The invention provides cells and methods of circularizing linear
DNA molecules. The cell is an isolated Escherichia coli cell which
transiently expresses the Cre recombinase protein from an
integrated Cre recombinase gene, and which is at least transiently
repressed for RecBCD activity. The cells are used in a method of
circularizing a linear DNA molecule comprising at least two loxP
sites. The DNA molecule is introduced into the cells, and the
linear DNA molecule is joined at said loxp sites.
Inventors: |
Sorge; Joseph A.; (Del Mar,
CA) ; Carstens; Carsten-Peter; (San Diego,
CA) |
Correspondence
Address: |
EDWARDS ANGELL PALMERS & DODGE LLP
P.O. Box 55874
Boston
MA
02205
US
|
Assignee: |
Stratagene California
|
Family ID: |
46323217 |
Appl. No.: |
11/284343 |
Filed: |
November 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10057050 |
Jan 25, 2002 |
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11284343 |
Nov 21, 2005 |
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09513710 |
Feb 25, 2000 |
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10057050 |
Jan 25, 2002 |
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Current U.S.
Class: |
435/91.1 ;
435/252.33; 435/488 |
Current CPC
Class: |
C12N 15/66 20130101;
C12N 15/10 20130101; C12N 15/64 20130101; C12P 19/34 20130101; C12N
9/00 20130101 |
Class at
Publication: |
435/091.1 ;
435/488; 435/252.33 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C12N 15/74 20060101 C12N015/74; C12P 19/34 20060101
C12P019/34 |
Claims
1. An isolated Escherichia coli cell which transiently expresses
the Cre recombinase protein from an integrated Cre recombinase
gene, and which is at least transiently repressed for RecBCD
activity.
2. The cell of claim 1, lacking a functional recB gene.
3. The cell of claim 1, lacking a functional recC gene.
4. The cell of claim 1, wherein said RecBCD activity is transiently
repressed
5. The cell of claim 1, which inducibly expresses an inhibitor of
RecBCD, selected from the group consisting of lambda Gam, T3 gene
5.9 protein, and P22 Abc2 protein.
6. The cell of claim 1, which inducibly expresses said Cre
recombinase protein.
7. The cell of claim 1, which is competent.
8. The cell of claim 8, wherein said Cre recombinase is transiently
expressed by a process comprising: treating said cell to induce
expression of said Cre recombinase prior to making said cells
competent.
9. The cell of claim 1, wherein said integrated Cre recombinase
gene is operably linked to the arabinose promoter
10. A method of circularizing a linear DNA molecule comprising at
least two loxP sites, comprising introducing said DNA into said
cells of claim 1, wherein said linear DNA molecule is joined at
said loxP sites.
11. The method of claim 10, wherein said cell lacks a functional
recB gene.
12. The method of claim 10, wherein said cell lacks a functional
recC gene.
13. The method of claim 10, wherein said RecBCD activity of said
cell is transiently repressed.
14. The method of claim 10, wherein said cell inducibly expresses
an inhibitor of RecBCD selected from the group consisting of lambda
Gam, T3 gene 5.9 protein, and P22 Abc2 protein.
15. The method of claim 10, wherein said cell inducibly expresses
said Cre recombinase protein.
16. The method of claim 10, wherein said cell is competent.
17. The cell of claim 16, wherein said Cre recombinase is
transiently expressed by a process comprising: treating said cell
to induce expression of said Cre recombinase prior to making said
cells competent.
18. The cell of claim 1, wherein said integrated Cre recombinase
gene is operably linked to the arabinose promoter.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/057,050, filed Jan. 25, 2002, which is a
continuation of U.S. patent application Ser. No. 09/513,710, filed
Feb. 25, 2000. The entire teachings of these disclosures are
incorporated herein by reference.
TECHNICAL FIELD
[0002] The invention relates to methods, cells and kits useful for
circularizing linear DNA molecules.
BACKGROUND OF THE INVENTION
[0003] Circularization of linear nucleic acid molecules often
requires enzymatic steps. One alternative to circularizing linear
DNA molecules is by site-specific recombination. A number of
approaches for the joining of DNA molecules have been described
using the Cre-loxP site-specific recombination system, including
Sauer and Henderson, (1988), Gene 70, 331-341; WO 00/26396; WO
02/083889; and U.S. Pat. App. No. 2004/0092016.
SUMMARY OF THE INVENTION
[0004] It is an object of the invention to provide methods,
compositions and microorganisms useful in the circularization of
linear DNA molecules. This and other objects of the invention are
provided by one or more of the embodiments described below.
[0005] In one aspect, microorganisms for biotechnology applications
are provided. The microorganism is an isolated Escherichia coli
cell which transiently expresses the Cre recombinase protein from
an integrated Cre recombinase gene, and which is at least
transiently repressed for RecBCD activity.
[0006] In another aspect, the present invention provides for a
method of circularizing a linear DNA molecule comprising at least
two loxP sites. The method comprises introducing the linear DNA
molecule into an isolated Escherichia coli cell which transiently
expresses the Cre recombinase protein from an integrated Cre
recombinase gene, and which is at least transiently repressed for
RecBCD activity. The linear DNA molecule is joined at the loxP
sites.
[0007] In yet another aspect, the present invention provides for a
kit for the circularization of a linear DNA molecule, comprising
the cells described herein, and optionally an instruction manual
and packaging material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows the non-directional covalent joining of an
insert molecule with 5'-OH groups on each end to a right vector arm
and a left vector arm each comprising a topoisomerase polypeptide
on one end only.
[0009] FIG. 2 shows the directional covalent joining of an insert
molecule with a 5'-OH group on one end and a 5'-phosphate group on
the other end to a right vector arm and a left vector arm each
comprising a topoisomerase polypeptide on one end only.
[0010] FIG. 3 shows the directional covalent joining of an insert
molecule with a 5'-OH group on one end and a 5'-phosphate group on
the other end to a left vector arm comprising a topoisomerase
polypeptide on one end only and a right vector arm comprising a
ligation substrate site on one end.
[0011] FIG. 4 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to a linear vector molecule. The linear vector molecule
comprises a topoisomerase molecule on one end only and a ligation
substrate site on the other end.
[0012] FIG. 5 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, cos, on the other end.
[0013] FIG. 6 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, cos, on the other
end.
[0014] FIG. 7 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, LIC, on the other end.
[0015] FIG. 8 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, LIC, on the other
end.
[0016] FIG. 9 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only of a lambda vector arm.
[0017] FIG. 10 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right plasmid arm and a
left plasmid arm each comprising a topoisomerase polypeptide on one
end only.
[0018] FIG. 11 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only.
[0019] FIG. 12 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, a loxP site, on the other
end.
[0020] FIG. 13 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, a loxP site, on the
other end.
[0021] FIG. 14 shows the non-directional cloning of an insert
molecule with 5'-OH groups on each end to a right vector arm and a
left vector arm each comprising a topoisomerase polypeptide on one
end only and a cloning substrate site, a site for homologous
recombination, on the other end.
[0022] FIG. 15 shows the directional cloning of an insert molecule
with a 5'-OH group on one end and a 5'-phosphate group on the other
end to vector molecules comprising a topoisomerase polypeptide on
one end only and a cloning substrate site, a site for homologous
recombination, on the other end.
[0023] FIG. 16 shows a strategy of assembly of the targeting vector
using overlap extension PCR and the Ara operon site that is
targeted for recombination. Reamplification with the outer primers
results in 78 and 86 bp homology regions for recombination.
[0024] FIG. 17 shows inefficient isolation of loxP-containing
plasmid DNA from cells that express high levels of cre recombinase.
LoxP containing tester plasmids pKSL-Cam and pBA2.2 and the control
plasmid pUC18 (no loxP) were introduced into Sure, Sure:cre (with
pACYC-cre) or clones sure:ara-cre #1 to 3 of Sure containing an
integration replacement of AraBAD with the cre recombinase gene
(see Chapter 3.1). Miniprep DNA was isolated from individual
colonies and a 4 ul aliquot was digested with PvuII and separated
on a 1% Agarose gel. Unrearranged pKSL-Cam results in fragment s of
2513 bp, 1106 bp and 526 bp in length. After cre mediated deletion
of the Cam marker Pvull digestion results in 2513 bp and 413 bp
fragments. Assignment of Plasmid DNA and host to the lanes is shown
on the insert.
[0025] FIG. 18 shows undetectable expression of Cre in uninduced
sure 3.2.5 (GTG). PvuII digest of plasmid extracted from Sure:cre
(with pACYC-cre), Sure 3.2.5.(ara:cre V3.0 GTG) and R2V4.0 (clone
17 XL10 replacement Gold (ara:cre myc-tagged with AUG start, this
clone won't be mentioned further and just served as a control in
this experiment). Lane assignment as indicated by the inserted
key.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0026] As used herein, the term "join" or "joining" refers to both
covalent and noncovalent attachment of one nucleic acid to another,
or one end of a nucleic acid to another end of a nucleic acid.
"Covalent" joining refers to the attachment of one end of a nucleic
acid strand to another end of a nucleic acid strand via a phosphate
bond or to attachment of one end of a double-stranded nucleic acid
to another double-stranded end via phosphate bonding on one or both
strands. "Non-covalent" joining refers to attachment of one end of
a nucleic acid to another end via annealing of a single-stranded
region to each other; that is, no phosphate bond is generated in
this embodiment.
[0027] "Ligate" or "ligated" refers to the covalent joining of two
ends of one or more nucleic acid molecules.
[0028] "Complementary annealing" refers to annealing, or the
pairing of bases, of complementary regions of one or more nucleic
acids, and thus to the formation of hydrogen bonds and other
non-covalent interactions between pairs of bases.
[0029] A "topoisomerase" is a polypeptide that is capable of
covalently joining to at least one strand of a nucleic acid
molecule and ligating that strand to another strand, as described
hereinbelow. Topoisomerase according to the invention comprises
type I topoisomerases.
[0030] "Bound to" refers to a covalent bonding of a topoisomerase
polypeptide to a nucleic acid molecule.
[0031] "Nucleic acid molecule" refers to a double-stranded nucleic
acid, unless otherwise specified.
[0032] "One end only" refers to the presence of a topoisomerase
polypeptide at one end of a nucleic acid molecule, where the
nucleic acid molecule contains two ends.
[0033] The term "site" is meant to designate a contiguous stretch
of nucleotides, e.g., 1-100 bases in length, usually 5-25 bases in
length, e.g., 8-16 bases, that is susceptible to (i.e., a substrate
for) modification by an enzyme that modifies nucleic acids, e.g., a
ligase or a restriction enzyme.
[0034] A "cloning substrate site", as used herein, is a site
occurring on a nucleic acid molecule for the covalent or
non-covalent joining of nucleic acid sequences or for
recombination. Examples of cloning substrate sites include cos
sites, LIC sites, sites for site-specific recombination, such as
lambda attachment elements or loxP sites, sites for homologous
recombination, and ligation substrate sites.
[0035] A "ligation substrate site", as used herein, is a site
occurring on a nucleic acid molecule of the invention that is
capable of becoming covalently joined to another nucleic acid
molecule in the presence of a ligase enzyme, such as DNA
ligase.
[0036] A "vector arm" or a "linear arm", as used herein, is a
linear nucleic acid molecule, and is preferably a portion or
fragment of a bacteriophage or plasmid genome.
[0037] "Directional" cloning refers to a cloning method in which,
by selecting steps in the method, one can obtain a desired
orientation of a given nucleic acid molecule upon cloning into
another nucleic acid molecule or between two other nucleic acid
molecules; as used herein, "orientation" may refer to 5' to 3' with
reference to a given open reading frame or a given control region
or a known sequence. Thus, for example, an insert molecule may
contain an open reading frame having a 5'-3' orientation with
respect to transcription and the insert molecule may be
directionally cloned between a left and right vector arms such that
the ligated (cloned) molecule comprises, from 5' to 3': left vector
arm, 5' insert 3', right vector arm. "Non-directional" cloning
refers to cloning methods which produce a ligated molecule in which
the insert, for example, appears between the two arms in either
orientation.
[0038] As used herein, a genetic construct which is "integrated"
refers to a construct which has been inserted into the genome of
the bacterium (i.e., into the bacterial chromosome).
[0039] As used herein, an "inducible" expression system refers to
any expression system in which the transcription level can be
modulated by at least 2 fold (for example, at least 3 fold, 5 fold,
10 fold, 15 fold, 20 fold, 30 fold, 100 fold or more). As used
herein, an "inducible" expression system encompasses expression
systems which can be induced, for example, by an inducer chemical
(e.g., arabinose, lactose, IPTG, etc), a stimulus (e.g., heat,
cold, etc.), or growth conditions (e.g., cell density, medium pH,
etc). An "inducible" expression system also encompasses any
expression system which can be down-regulated, for example,
expression systems which can be down-regulated upon addition of a
chemical (e.g., glucose-repressible), environmental stimulus, and
the like.
[0040] As used herein, a cell is said to "transiently express" the
Cre recombinase protein when the presence of Cre is transient, or
when the abundance of Cre varies depending on the applied
condition. Ideally, the Cre recombinase protein is present in the
cell at the moment that the linear DNA molecule is introduced, and
substantially reduced (i.e., at least 2-fold reduced, for example,
3 fold, 5 fold, 10 fold, 20 fold, 100 fold reduced or more) in
abundance or absent during subsequent plasmid purification, for
example as measured using methods described in Kaczmarczyk &
Green (2003) Nucl. Acid Res. 31, e86. As used herein, a cell that
"transiently expresses" the Cre recombinase also contemplates a
cell in which the expression is never totally abolished. Therefore,
even if the protein is present at all times, a cell is said to
"transiently express" Cre if the expression levels can be
manipulated (e.g., by addition of an inducer) such that its
abundance is significantly higher (e.g., at least 2-fold higher,
for example, 3 fold, 5 fold, 10 fold, 20 fold, 100 fold higher or
more) at one point (e.g., at the point that the linear DNA molecule
is introduced) than at another (e.g., when cells are collected for
plasmid purification). One can "transiently express" the Cre
recombinase that is expressed at a low, basal level, for example,
by addition of an inducer. Alternatively, a repressor can be
removed to achieve transient expression.
[0041] As used herein, a cell is "transiently repressed" for RecBCD
activity when the ATP-dependent nuclease activity of the RecBCD
enzyme is substantially reduced (i.e., at least 50% up to 100%
reduced, for example 60%, 70%, 75%, 80%, 90%, 95%, 99% up to 100%)
during the time that the linear DNA molecule is introduced to the
cell, when compared with a wild-type cell, and as measured, for
example, as described in Boehmer & Emmerson, (1991) Gene 102,
1. For example, the detectable ATP-dependent nuclease activity of
the RecBCD enzyme can be repressed transiently by, for example by
replacing its promoter with one that can be repressed by selected
stimuli, whether environmental, chemical or otherwise, and adding
the repressor to the growth medium to repress its expression. In
addition, a cell that transiently expresses an inhibitor of the
ATP-dependent nuclease activity of the RecBCD enzyme is also said
to be "transiently repressed" for RecBCD activity. ATP-dependent
nuclease activity of RecBCD can be measured in vitro as described
in the art (e.g., Boehmer & Emmerson, (1991) Gene 102, 1). As
used herein, a cell that is "at least transiently repressed" for
RecBCD activity encompasses both a cell that is "transiently
repressed" for RecBCD activity, as well a cell that has
continuously reduced or abolished RecBCD activity when compared
with a wild-type cell, for example, as a result of a mutation
within the recB, recC or recD gene.
Insert Polynucleotide Molecules
[0042] Insert polynucleotide molecules comprise isolated and
purified double-stranded DNA, double-stranded RNA, or
double-stranded DNA/RNA hybrid nucleic acids. An insert molecule
can be a full-length molecule or a fragment of a full-length
molecule. Further, an insert molecule can be naturally-occurring,
i.e., found in nature or recombinant.
[0043] Preferably, insert polynucleotides are isolated free of
other components, such as proteins and lipids. Insert
polynucleotides can be made by a cell and isolated or can be
synthesized in the laboratory, for example, using an automatic
synthesizer or an amplification method such as PCR. Where an insert
polynucleotide is prepared by PCR, the insert is generated using a
pair of primers comprising a 3'-primer and a 5'-primer. Both the
3'-primer and the 5'-primer can comprise a 5'-hydroxyl group to
produce an insert with 5'-hydroxyl groups (5'-OH) on both ends.
Alternatively, one of the primers can comprise a 5'-hydroxyl group
and one can comprise a 5'-phosphate group to produce an insert with
a 5'-OH group on one end and a 5'-phosphate (5'-P) group on the
other end. Optionally, both the 3'-primer and the 5'-primer can
comprise a 5'-phosphate group to produce an insert with 5'-P groups
on both ends.
Molecules Flanking an Insert Molecule
[0044] An insert polynucleotide molecule can be covalently joined
to several types of molecules, such as a double-stranded DNA, a
double-stranded RNA, and a double-stranded DNA/RNA hybrid molecule.
Preferably, an insert polynucleotide molecule is covalently joined
to a vector molecule or to vector molecules such as a linear arm of
a plasmid or bacteriophage. Vectors suitable for ligation of an
insert molecule include bacteriophage, such as bacteriophage
lambda, including, but not limited to lambda insertion vectors such
as Lambda ZAP.RTM.II vector, ZAP Express.RTM. vector, Lambda
ZAP.RTM.-CMV vector (Stratagene), lambda gt10, and lambda gt11.
Lambda replacement vectors, for example Lambda FIX.RTM.II vector,
Lambda DASH.RTM.II vector, and Lambda EMBL3 and Lambda EMBL4
(Stratagene) can also be used as vectors.
[0045] Both prokaryotic and eukaryotic linear plasmids can be used
as vectors. See e.g., Meinhardt et al. (1997) Appl. Microbiol.
Biotechnol. 47:329-36; Fukuhara, (1995) FEMS Microbiol.
Lett.131:1-9; Hinnebusch & Tilly, (1993) Mol Microbiol.
10:917-22. For example, the plasmid prophage N15 of E. coli is a
suitable linear plasmid vector. See Rybchin & Svarchevsky
(1999) Mol. Microbiol. 33:895-903.
[0046] Vector nucleic acid polynucleotides, such as bacteriophage
and plasmids can be isolated and purified from cells carrying these
elements according to methods well known in the art. See e.g.
MOLECULAR CLONING: A LABORATORY MANUAL (Sambrook et al., eds., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) and
Ausubel (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al.,
eds., John Wiley & Sons, New York, 1987)). Additionally, many
bacteriophage and plasmid vectors are commercially available. The
bacteriophage or plasmid nucleic acid can be prepared, if necessary
by cleavage with an appropriate restriction enzyme such that the
digested bacteriophage or plasmid nucleic acid is compatible with
an insert molecule.
[0047] Preferably, an insert molecule is covalently joined to right
and left lambda linear vector arms such that the insert molecule is
positioned between right and left lambda linear vector arms. In
lambda insertion vectors, a left vector arm can comprise lambda
nucleic acids occurring to the left of the insertion site and a
right vector arm can comprises lambda nucleic acids occurring to
the right of the insertion. In lambda replacement vectors, a left
lambda arm comprises lambda nucleic acid occurring to the left of
the nucleic acids to be replaced by the insert nucleic acids and a
right lambda arm comprises lambda nucleic acids occurring to the
right of the nucleic acids to replaced by the insert nucleic acids.
Lambda vectors can vary in nucleic acid sequence; however, the left
arm can typically comprise the head and tail genes A-J, while the
right arm can typically comprise from PR through a cosR site of a
lambda genome.
[0048] Preferably, the vector or flanking molecule to which the
insert is to be covalently joined is a linear molecule comprising a
topoisomerase covalently linked to only one end of the linear
molecule. A double-stranded DNA, double-stranded RNA, or
double-stranded DNA/RNA molecule with one topoisomerase molecule
bound to one end of the DNA or RNA molecule is a univalent
molecule. DNA topoisomerases catalyze a conversion in the linking
number of a double-stranded DNA molecule. The linking number is the
number of times one DNA strand crosses over the second DNA strand
in space. Type 1 topoisomerases act by making a transient break in
one strand of a nucleic acid. A type 1 topoisomerase first binds to
a nucleic acid and nicks one strand of the nucleic acid. A stable
complex is formed where the free 3'-phosphate end of the nicked
strand is covalently bound to a tyrosine residue of the enzyme. The
second strand is pulled through the gap in the first strand and the
gap is then sealed by the enzyme. The gap can be sealed at the same
bond originally nicked or the complex can combine with a
heterologous nucleic acid, such as an insert molecule, that has a
5'-hydroxy end. Where the complex is combined with a heterologous
nucleic acid, a recombinant molecule is formed.
[0049] Type 1 topoisomerases include, but are not limited to E.
coli topoisomerase I (Keck et al., (1999) Nat. Strut. Biol. 6:900),
E. coli topoisomerase III (Mondragon et al., (1999) Structure Fold.
Des. 7:1373), S. cerevisiae topoisomerase III (Kim et al., (1992)
J. Biol. Chem. 267:17178), human topoisomerase III (Hanai et al.,
(1996) Proc. Natl. Acad. Sci. 93:3653), the type I topoisomerase
from chloroplasts (Mukherjee et al. (1994) 269:3793; Fukata et al.
(1991) J. Biochem (Tokyo) 109:127), thermophilic reverse gyrases
(Nadal et al., (1994) J. Biol. Chem. 269:5255; Slesarev et al.,
(1991) J. Biol. Chem. 266:12321; Bouthier de la Tour et al., (1991)
J. Bact. 173:3921), thermophilic D. amylolyticus topoisomerase III
(Slesarev et al., (1991) J. Biol. Chem. 266:12321), and vaccinia
DNA topoisomerase I (Shuman et al., (1987) Proc. Natl. Acad. Sci.
84:7478). Site-specific type I DNA topoisomerases are particularly
useful in the invention. Site-specific type I DNA topoisomerases
include vaccinia topoisomerase and pox virus topoisomerases.
[0050] A topoisomerase enzyme can be covalently linked to a vector
or flanking molecule by, for example, the method of Heyman et al.
(Genome Res. (1999) 9:383). Briefly, Vaccinia DNA topoisomerase
cleaves the phosphodiester backbone of one strand of a nucleic acid
at a consensus pentopyrimidine element: 5'-C/TCCTT-3' (SEQ ID
NO:1). This element can be added onto the end of a vector or
flanking molecule. Vaccinia topoisomerase can then be incubated
with the vector or flanking molecule such that the topoisomerase
becomes covalently bound to the underlined T in the C/TCCTT
sequence. Optionally, nuclease treatment, such as exonuclease III
treatment can be used to remove single strand ends from the element
such that a blunt-ended insert fragment with topoisomerase bound to
the molecule is formed.
[0051] Optionally, the molecule to which the insert is to be
covalently joined is a linear molecule comprising a ligation
substrate site at a first end of the linear molecule. A ligation
substrate site comprises a site for nucleic acid ligation that is
mediated by a ligase enzyme. A ligation substrate site can comprise
any double-stranded nucleic acid that has blunt ends or protruding
termini that can be covalently joined to another nucleic acid
molecule in the presence of a ligase enzyme. Preferably, the
ligation substrate site comprises a 5'-phosphate group and is
complementary to one end of an insert molecule. A ligation
substrate site can be produced by, for example cleaving a
double-stranded nucleic acid molecule with a restriction enzyme
that produces blunt-ended termini, 5'- protruding ends, or
3'-protruding ends and purifying the nucleic acid molecule. A
ligation between a linear molecule comprising a ligation substrate
site and an insert molecule takes place in the presence of a ligase
enzyme such as bacteriophage T4 DNA ligase or Pfu DNA ligase
(Stratagene). Preferably, the vector or flanking molecule to which
the insert is to be covalently joined is a linear molecule
comprising a topoisomerase covalently linked to only one end of the
molecule or a ligation substrate site at one end of the linear
molecule. The second end of the linear molecule preferably
comprises a cloning substrate site such as, a cos site, a LIC site,
a site-specific recombination site (such as a loxP site or lambda
attachment element), a homologous recombination site or a ligation
substrate site.
[0052] A bacteriophage lambda genome has cos sites at the ends of
the genome. See, LAMBDA II (Roger W. Hendrix, ed., Cold Spring
Harbor Laboratory Press) 1983; Higgins et al., (1995) J. Mol. Biol.
252:31; Higgins et al., (1994) EMBO J. 13:6152; Cue et al., (1993)
J. Mol. Biol. 234:594; Cue et al., (1993) Proc. Natl. Acad. Sci.
USA 90:9290. Cleavage occurs at a left cos site (as defined on a
standard lambda map) to generate a free end that is inserted into a
capsid. The insertion of nucleic acid continues until a right cos
site is encountered. Cleavage occurs at the right cos site to
generate the second end. Any nucleic acid molecule that is
contained between two cos sites can be packaged. A nucleic acid
molecule comprising a cos site, a fragment of a cos site, a mutant
of a cos site, or a variant of a cos site can be isolated from a
preparation of bacteriophage lambda DNA or may synthesized in the
laboratory. A nucleic molecule comprising a cos site can be ligated
to the end of the molecule to which the insert is to be covalently
joined. Alternatively, a cos site can be added to the end of a
molecule to which the insert is to be covalently joined using
standard molecular biology cloning techniques such as PCR. In the
methods of the invention distal ends (i.e., the ends of vector arms
not covalently joined to an insert molecule) of vector arms
containing terminal cos sites can be readily annealed to one
another in E. coli host cells by virtue of their explicit sequence.
cos sites do not appreciably anneal in vitro at room
temperature.
[0053] A ligation-independent cloning (LIC) site can be any size,
but is preferably 12 to 13 nucleotides or longer. Sites longer than
12-13 nucleotides may work more efficiently, e.g., up to 24 bases,
or up to 48 bases or longer. See Aslanidis and de Jong, (1990)
Nucleic Acids Res. 18:6069. The 12-13 nucleotide terminus can
comprise any nucleic acid sequence; however, preferably one or none
of the nucleotides of a 3' strand of the 12-13 nucleotide terminus
is an adenosine. A nucleic molecule comprising a LIC site can be
ligated to the ends of the vector or flanking molecule to which the
insert is to be covalently joined. Alternatively, a LIC site can be
added to the end of a vector or flanking molecule to which the
insert is to be covalently joined using standard molecular biology
cloning techniques, such as by PCR.
[0054] Where the second end of a linear molecule comprises a LIC
site, a ligated insert/vector molecule will be formed that
comprises LIC ends at each end of the ligated insert/vector
molecule. The insert can then be joined to a LIC ready vector.
Aslanidis et al., (1994) PCR Methods Appl. 4:172; Aslanidis and de
Jong (1990) Nucleic Acids Res. 18:6069. Briefly, the ligated
insert/vector molecule is subjected to treatment with, for example,
Pfu DNA polymerase in the presence of dATP. In the absence of dTTP,
dGTP, and dCTP, the 3'- to 5'- exonuclease activity of Pfu DNA
polymerase removes 12 to 13 nucleic acids from the 3'-ends of the
ligated insert/vector molecule. This activity continues until the
first adenine is encountered. This produces a ligated insert/vector
molecule with 5 '-extended single-stranded tails that are
complementary to the single-stranded tails of a LIC ready vector.
The ligated insert/vector molecule will anneal to the LIC ready
vector without further enzymatic treatment.
[0055] The second end of the linear molecule can further comprise a
site for homologous recombination or a site for site-specific
recombination. Homologous recombination is a recombination event
occurring between homologous sequences of nucleic acids. The
enzymes responsible for homologous recombination can use any pair
of homologous sequences as substrates, although some types of
nucleic acid sequences can be favored over others. Sites for
homologous recombination comprise nucleic acid sequences that are
homologous to the nucleic acid sequences of a cloning vector, such
as a circular plasmid. The sites can insert (or integrate) into a
cloning vector by homologous recombination, thereby inserting or
displacing a nucleic acid sequence, or deleting a nucleic acid
sequence altogether.
[0056] To create a homologous recombinant plasmid cloning vector, a
plasmid cloning vector is prepared which contains homologous
recombination nucleic acid sites that are substantially homologous
to those sites occurring on the ligated insert/vector of interest.
Substantially homologous nucleic acid sequences are those nucleic
acid sequences that share sufficient nucleic acid sequence homology
to provide for sufficient homologous recombination between a
ligated insert/vector sequence and a plasmid cloning vector.
Sufficient nucleic acid sequence homology is the amount which
provides for homologous recombination at a frequency which allows
for detection of plasmid cloning vectors in which homologous
recombination and integration of the ligated vector/insert has
occurred. Substantially homologous nucleic acid sequences
preferably share regions with about 60% to 100% nucleic acid
sequence homology, and more preferably about 75% to 100% homology
in the nucleic acid sequence. A site for homologous recombination
can be present in the plasmid cloning vector in two or more copies.
The homologous recombination sites in the plasmid cloning vector
are of sufficient length for successful homologous recombination
with a ligated insert/vector molecule. Typically, each homologous
recombination site is at least 30, 75, 100, 150, 250, 500, or 1000
base pairs. The ligated insert/vector sequence comprises these
substantially homologous recombination sites at both the 5'- and
3-' ends. The ligated insert/vector sequence is transformed into a
host cell, such as an E. coli cell that contains the plasmid
cloning vector. Preferably, the host cell is RecA+. RecA is the
product of the recA locus of E. coli and is a protein that is
involved in recombination.
[0057] In addition to homologous recombination as described above,
enzyme-assisted site-specific integration systems are known in the
art and can be applied to integrate a ligated nucleic acid
insert/vector molecule at a predetermined location in a cloning
vector molecule. Site-specific recombination is a recombination
event between specific pairs or sequences. The recombination event
involves specific sequences of nucleic acids comprising a short
stretch of homology necessary for the recombination event. The
enzymes involved in the recombination event will act only on this
particular pair of target sequences. Examples of such
enzyme-assisted integration systems include the Cre
recombinase/loxP target system (e.g., as described in Baubonis and
Sauer (1993) Nucl. Acids Res. 21:2025; and Fukushige and Sauer,
(1992) Proc. Natl. Acad. Sci. USA 89:7905). A loxP site (locus of
crossing over) comprises two 13 base pair inverted repeats
separated by an 8 base pair asymmetric spacer region:
TABLE-US-00001 ATAACTTCGTATA ATGTATGC TATACGAAGTTAT (SEQ ID
NO:2)
[0058] A loxP site of the invention comprises variants and mutants
of this sequence that function to produce site-specific
recombination. Cre is a 38 kDa recombinase protein from
bacteriophage P1 which mediates intramolecular and intermolecular
site-specific recombination between loxP sites. Sauer, (1993)
Methods Enzymol. 225:890. A loxP site is an asymmetrical nucleotide
sequence and two lox sites on the same DNA molecule can have the
same or opposite orientation with respect to one another. See U.S.
Pat. No. 4,959,317. Where two loxP sites occur in the same
orientation on a nucleic acid molecule, recombination between the
loxP sites results in the deletion of the nucleic acid segment
located between the two loxP sites and a connection between the
resulting ends of the original nucleic acid molecule. The deleted
nucleic acid molecule will form a circular molecule of nucleic
acid. The original nucleic acid molecule and the circular nucleic
acid molecule will each contain a single loxP site. Where two loxP
sites occur in opposite orientations on the same nucleic acid
molecule recombination will result in an inversion of the
nucleotide sequence of the nucleic acid segment located between the
two loxP sites. Further, where two loxP sites occur on each of two
nucleic acid segments, reciprocal exchange of nucleic acid segments
proximate to the loxP sites can occur.
Methods of Covalently Joining
[0059] Insert polynucleotide molecules comprising a 5'-OH group on
each end or a 5'-OH on one end and a 5'-phosphate group on the
other end can be covalently joined to flanking polynucleotide
molecules such that non-directional or directional covalent joining
is achieved. Where an insert polynucleotide molecule has a 5'-OH
group on each end non-directional covalent joining of the insert to
flanking polynucleotide molecules results. For example, where an
insert polynucleotide (I) with a 5'-OH group at each end is
covalently joined to flanking molecules, for example, a left vector
arm (LVA) and a right vector arm (RVA) each with a topoisomerase
polypeptide covalently joined at only one end, the result will be
non-directional covalent joining of the molecules. A LVA, RVA, and
insert molecule are incubated together under conditions sufficient
to permit their topoisomerase-mediated covalent joining to form a
covalently joined nucleic acid molecule where the insert molecule
is positioned between the LVA and RVA. Four different covalently
joined products (ligated insert/vector molecules) will result:
LVA-I-LVA, RVA-I-RVA, LVA-I-RVA, and RVA-I-LVA. Only the LVA-I-RVA
and RVA-I-LVA products are viable replication competent entities.
Where an insert polynucleotide has a 5'-OH group on one end and a
5'-phosphate group on the other end directional covalent joining of
the insert to flanking polynucleotide molecules can result. For
example, where an insert polynucleotide is covalently joined to a
flanking molecules such as a LVA and a RVA, each comprising a
topoisomerase covalently bound to only one end, directional
covalent joining of the molecules can result. A first vector arm,
for example, a LVA is covalently joined to an insert molecule at
the 5'-OH end by incubating a LVA and an insert molecule together
under conditions sufficient to permit topoisomerase-mediated
covalent joining to form a ligated nucleic acid molecule where the
insert molecule is positioned adjacent to a LVA to create
LVA-I-phosphate. The 5'-phosphate end of the insert is unable to be
ligated to the LVA or RVA because either the LVA or RVA has a
3'-phosphate, which is the site to which a topoisomerase
polypeptide is joined to the LVA and RVA. The LVA-I-5'-phosphate is
treated with phosphatase, under conditions which permit removal of
a 5'-phosphate group from the ligated nucleic acid resulting in a
LVA-I-5'-OH molecule. The LVA-I-5'-OH molecule is then covalently
joined to the RVA to form LVA-I-RVA by incubating a LVA-I-5'-OH
molecule with a RVA under conditions which permit topoisomerase
covalent joining to form a ligated molecule where the insert
molecule is positioned between a RVA and a LVA (a ligated
insert/vector molecule).
[0060] Alternatively, an insert polynucleotide comprising a 5'-OH
group on one end and a 5'-phosphate group on the other end can be
covalently joined in a directional manner to a flanking nucleic
acid molecule comprising a topoisomerase polynucleotide on only one
end and to a second flanking molecule comprising a ligation
substrate site on one end. For example, an insert molecule can be
covalently joined to a flanking nucleic acid molecule, such as a
LVA, comprising a topoisomerase polypeptide on only one end and to,
for example, a RVA comprising a ligation substrate end on one end.
The insert LVA, and RVA are covalently joined by
topoisomerase-mediated joining and ligase-mediated joining under
conditions sufficient to form a ligated nucleic acid where the
insert molecule is positioned between a LVA and a RVA to form
LVA-I-RVA (a ligated insert/vector molecule). This reaction can
take place in one step.
[0061] After the ligated insert/vector molecule described above has
been constructed, the two vector arms can be non-covalently or
covalently joined to one another, at the ends distal to the
covalently attached topoisomerase polypeptide or ligation substrate
site (i.e., at their free ends), by a number of methods such that a
circular molecule is formed. For example, the ends of the ligated
insert/vector molecule can comprise ligase substrate sites or
complementary nucleic acid sequences such that the ends can be
joined by ligase enzyme mediated ligation or complementary sequence
annealing. Further, where the ends of the ligated insert/vector
molecule comprise 5'-OH groups the ends can be joined by
topoisomerase mediated ligation using a polynucleotide comprising a
topoisomerase polypeptide at both ends of the polynucleotides. See
e.g. U.S. Pat. No. 5,766,891. Further, where the ends of the
ligated insert/vector molecule comprise in vitro or in vivo
site-specific recombination sites or in vivo homologous
recombination sites the ligated insert/vector molecule can be
recombined into a circular plasmid containing the same
recombination sites.
[0062] The methods of directional and non-directional covalently
joining of nucleic acid molecules are useful in, for example,
end-labeling, ligand tagging, and molecular cloning.
[0063] Methods of Molecular Cloning Insert polynucleotide molecules
comprising a 5'-OH group on each end or a 5'-OH on one end and a
5'-phosphate group on the other end can be cloned into vector
molecules such that non-directional or directional cloning is
achieved.
[0064] Non-directional cloning can be accomplished by cloning a
polynucleotide insert molecule comprising 5'-OH groups at both ends
of the molecule into a nucleic acid vector. For example, an insert
polynucleotide (I) with a 5'OH group at each end can be cloned into
a vector, such as a left vector arm (LVA) and a right vector arm
(RVA) where each vector arm has a topoisomerase polypeptide
covalently joined at only one end of the vector arm. The result
will be non-directional covalent joining of the molecules.
Preferably, the LVA and RVA molecules have a cloning substrate
site, such as a cos site, a LIC site, a loxP site, a site for
homologous recombination, a site for site-specific recombination,
or a ligase substrate site at the other end of the molecule. A LVA,
RVA, and insert molecule are incubated together under conditions
sufficient for topoisomerase-mediated covalent joining of the
molecules to form a ligated nucleic acid wherein the insert
molecule is positioned between the LVA and RVA. Four different
covalently joined products will result: LVA-I-LVA, RVA-I-RVA,
LVA-I-RVA, and RVA-I-LVA (ligated insert/vector molecules). Only
the LVA-I-RVA and RVA-I-LVA products are viable replication
competent entities.
[0065] Directional cloning can be accomplished by cloning a
polynucleotide insert molecule comprising a 5'-OH group at one end
of the molecule and a 5'-phosphate group at the other end into a
nucleic acid vector. For example, an insert polynucleotide (I) with
a 5'OH group at one end and a 5'-phosphate at the other end can be
cloned into a linear cloning vector, where the linear cloning
vector has a topoisomerase polypeptide covalently joined at one end
and a ligation substrate site at the other end. The insert
polynucleotide, the linear cloning vector, and a ligase are
incubated together under conditions sufficient for their covalent
joining to form a ligated circular vector (a ligated insert/vector
molecule). The circular vector can then be transformed into a host
cell.
[0066] Directional cloning can also be accomplished by cloning an
insert polynucleotide having a 5'-OH group on one end and a
5'-phosphate group on the other end into a vector where the vector
comprises, for example, two vector arm molecules comprising a
topoisomerase polynucleotide at only one end and a cloning
substrate site at the other end. For example, a first vector arm,
LVA, is covalently joined to an insert molecule at the 5'-OH end by
incubating a LVA and an insert molecule together under conditions
sufficient to permit topoisomerase-mediated covalent joining to
form a ligated nucleic acid molecule where the insert molecule is
positioned adjacent to a LVA to create LVA-I-phosphate. The
5'-phosphate end of the insert is unable to be ligated to the LVA
or RVA because a topoisomerase polypeptide is joined to the LVA and
RVA at the 5'-phosphate. The LVA-I-5'-phosphate is treated with
phosphatase, under conditions which permit removal of a
5'-phosphate group from the ligated nucleic acid resulting in a
LVA-I-5'-OH molecule. The LVA-I-5'-OH molecule is then covalently
joined to the RVA to form LVA-I-RVA by incubating a LVA-I-5'-OH
molecule with a RVA under conditions which permit topoisomerase
covalent joining to form a ligated molecule where the insert
molecule is positioned between a RVA and a LVA (a ligated
insert/vector molecule). Preferably, the cloning substrate site is
a cos site, a LIC site, a loxP site, a site for homologous
recombination, a site for site-specific recombination, or a
ligation substrate site.
[0067] Alternatively, directional cloning can be accomplished with
an insert polynucleotide comprising a 5'-OH group on one end and a
5'-phosphate group on the other end and two vector molecules. One
vector molecule comprises a topoisomerase polynucleotide on only
one end and a cloning substrate site on the other end. The other
vector molecule comprises a ligation substrate site on one end and
a cloning substrate site on the other end. An insert, a first
vector molecule comprising a topoisomerase polypeptide at one end
and a cloning substrate site at the other end, such as a LVA, and a
second vector molecule such as a RVA comprising a ligation
substrate site at one end and a cloning substrate at the other end
are covalently joined by topoisomerase-mediated joining and
ligase-mediated joining under conditions sufficient to form a
ligated nucleic acid where the insert molecule is positioned
between the LVA and the RVA vector molecules (a ligated
insert/vector molecule). Preferably, the cloning substrate site is
a cos site, a LIC site, a loxP site, a site for homologous
recombination, a site for site-specific recombination, or a
ligation substrate site.
[0068] Where the ligated insert/vector molecule comprises cos sites
at each end, the linear molecule can be transformed directly into a
host cell. Where the ligated insert/vector molecule comprises LIC
ends at each end, the LIC ends can be annealed to a circular
plasmid vector with LIC compatible ends. The circular molecule can
be transformed into a host cell. Where the ligated insert/vector
molecule comprises loxP sites on both ends, the ligated
insert/vector molecule can be recombined into a circular plasmid in
vitro in the presence of Cre recombinase. The recombinant circular
plasmid can then be transformed into a host cell. Alternatively, a
ligated insert/vector molecule with loxP sites at both ends of the
molecule can be directly transformed into a host cell, such as E.
coli harboring a plasmid suitable for site-specific recombination.
The host cell may be rec A+or recA-, and is preferably recA-. Where
the covalently joined insert/vector molecule comprises sites for
homologous recombination at each end, the covalently joined
insert/vector molecule can be directly transformed into a suitable
host cell harboring a plasmid suitable for homologous
recombination.
[0069] The covalently joined insert/vector can be transformed into
a prokaryotic or eukaryotic cell. Preferably, the covalently joined
insert/vector is transformed into a prokaryotic host cell, such as
a bacteria cell such as E. coli. Transformation of a ligated
insert/vector molecule into a host cell can be done by any method
known in the art. Methods for transformation of host cells can be
found in Sambrook et al. and Ausubel and include, but are not
limited to transfection, chemical transformation, electroporation,
and lipofection. Where a bacteriophage lambda vector has been used
according to the invention, the ligated insert/lambda vector can be
packaged in vitro and then transfected into host cells, such as
XLI-Blue E. coli. See e.g. Sambrook et al.
Host Cells
[0070] In another aspect, bacterial cells useful for
circularization of linear DNA molecules are disclosed. The
bacterial cell is an isolated bacterial cell, for example a gram
negative bacterium, for example an isolated Escherichia coli cell,
which transiently expresses the Cre recombinase protein and which
is at least transiently repressed for RecBCD activity.
[0071] In one embodiment, the cell contains the coding sequence for
the Cre recombinase protein operably linked to an inducible
promoter. The Cre recombinase gene can be incorporated into the
bacterial chromosome (i.e., integrated), provided on a plasmid, or
both. In one embodiment, the cell transiently expresses the Cre
recombinase protein from an integrated construct. The inducible
promoter can be any known in the art, and include the galactose,
lactose, tac, arabinose, rhamnose, tet, or trp promoters.
[0072] In one embodiment, the inducible promoter is the an
arabinose-inducible promoter, for example the arabinose promoter of
E. coli. Expression systems using the arabinose operon from E. coli
has been well described (WO0173082 A2; Hirsh & Schleif, (1973)
J Mol Biol 80(3), 433-44; Hahn, et al. (1984) J Mol. Biol 180(1),
61-72; Kosiba & Schleif, (1982) Mol. Biol 156(1), 53-66, each
incorporated by reference). In another embodiment, the inducible
promoter is a modified arabinose promoter, that is mutated to
reduce basal levels of Cre protein expression. Modifications to
reduce basal expression levels of Cre include introducing mutations
into the araB promoter region, for example, the ribosome binding
site, replacement of the start codon from ATG to GTG, and
elimination by mutagenesis of cryptic start sites. Modifications of
this type are described further in Example 10.
[0073] In addition to its role in recombination, the RecBCD enzyme
is a nuclease that selectively hydrolyzes linear double-stranded
DNA (dsDNA) to deoxynucleotides. The reaction is ATP-dependent, and
does not affect closed circular supercoiled or nicked circular
dsDNAs. For example, the RecBCD enzyme, which plays a critical role
in recombination and repair of double-stranded DNA (dsDNA) breaks
(Kowalczykowski et al. (1994) Microbiol Rev 58: 401-465), is also a
major exonuclease activity (ExoV) which rapidly degrades linear DNA
molecules, thereby inhibiting certain types of recombination
(Telander-Muskavitch & Linn (1981) Enzymes 14A: 233-250).
Reduction or elimination of RecBCD activity, in particular the
ATP-dependent nuclease activity, which can result in reduced
recombination efficiency, is therefore preferred. Therefore, in one
embodiment, the cells useful for the circularization of linear DNA
molecules are at least transiently repressed in RecBCD activity.
Preferably, the cells are reduced in the ATP-dependent nuclease
activity of the RecBCD enzyme at the time the linear DNA molecule
is introduced into the cell. Therefore, in one embodiment, the cell
contains one or more mutations within the recB gene, the recC gene,
or both. In another embodiment, the RecBCD gene is driven by a
repressible promoter. In still another embodiment, the cell is
recBC-.
[0074] In yet another embodiment, the cell comprises an inducible
construct which encodes an inhibitor of the RecBCD enzyme, in
particular the ATP-dependent exonuclease activity of RecBCD.
Examples of such inhibitors include the lambda Gam, the P22 Abc2,
and T3-phage gene 5.9 product (See, for example, Poteete et al.
(1988) J Bacteriol. 170:2012-21; Marsic et al. (1993) J Bacteriol.
175:4738-43; Murphy, (1991) J Bacteriol. 173: 5808-5821). Such
inhibitors can be expressed at least transiently (i.e., either
constitutively or transiently). It is only important that the
inhibitor be present in the cell at the time the linear DNA is
introduced into the cell, in order to minimize degradation of
linear DNA at the time it is introduced into the cell. In one
embodiment, the inhibitor of the RecBCD enzyme is the lambda Gam.
In another embodiment, the inhibitor is the 5.9 gene product from
phage T3. Gam is a protein encoded by the lambda genome involved in
double strand break repair homologous recombination. Gam has been
shown inhibit cellular nuclease activity such as that encoded by
the RecBCD system of E. coli.
[0075] As previously noted, at the time the linear DNA molecule is
introduced to the cell, it is preferable that the cell contains or
expresses the Cre recombinase. Furthermore, reduction in the
ATP-dependent exonuclease activity of the RecBCD enzyme is
preferable. Therefore, in one embodiment, a host cell expresses
both the Cre recombinase and an inhibitor of the RecBCD enzyme
selected, for example, from the group consisting of lambda Gam, P22
Abc2, and T3-phage gene 5.9 product. According to this embodiment,
the Cre recombinase and the inhibitor are expressed using an
inducible promoter, for example in an integrated expression
construct. In one embodiment, the inducible promoter is an
arabinose-inducible promoter.
[0076] The cells described herein can be used for circularization
of linear pieces of DNA. The cells can be used to circularize a
single piece of DNA, containing two loxP sites. Alternatively, the
cells can be used to circularize a plurality of linear DNA
molecules, each containing at least two loxp sites. The only
requirement is that at least two directly repeating loxP sites be
present on each of the DNA molecules needing to be
circularized.
[0077] In another embodiment, the cells are competent for
transformation. Any of the well-established methods of generating
competent cells that are known in the art can be used. One method
involves growing cells to log phase or early stationary phase and
exposing the cells to CaCl.sub.2 at 0.degree. C. (see, e.g.,
Sambrook, et al., Molecular Cloning: a Laboratory Manual, 2nd
Edition, eds. Sambrook, et al., Cold Spring Harbor Laboratory
Press, (1989), incorporated herein by reference). Cells can be
contacted immediately with exogenous DNA or frozen in glycerol or
DMSO for subsequent use. Upon thawing to 4.degree. C. and
contacting with plasmid DNA, frozen competent cells typically have
transformation efficiencies of 1.times.10.sup.8-1.times.10.sup.11
transformants/.mu.g of plasmid DNA.
[0078] Electroporation has also been used to transform cells (see,
e.g., Dower et al., Nucleic Acids Research, 16: 6127-6145 (1988);
Taketo, Biochimica et Biophysica Acta, 949: 318-324 (1988); Chassy
and Flickinger, FEMS Microbiology Letters, 44: 173-177 (1987); and
Harlander, Streptococcal Genetics, eds. Ferretti and Curtiss,
American Society of Microbiology, Washington, D.C., pp. 229-233
(1987), all incorporated herein by reference). Electroporation
methods rely on creating temporary holes in cell membranes by
exposing cells to a high voltage electric impulse to facilitate the
uptake of exogenous nucleic acids (see, e.g., Andreason and Evans,
Biotechniques, 6: 650-660 (1988), incorporated herein by
reference). Cells exposed to an electroporation buffer (e.g.,
10-15% glycerol) are generally stored by freezing to provide a
supply of electrocompetent cells (see, e.g., U.S. Pat. No.
6,004,804, incorporated herein by reference in its entirety). Other
methods of preparing competent cells are described, without
limitation, in the Examples below.
[0079] In yet another embodiment, cells rendered competent for
transformation are prepared which contain or express the Cre
recombinase, and are repressed in RecBCD activity. As previously
noted, Cre recombinase expression can be induced, for example, by
means of an inducer if an inducible promoter is used, shortly prior
to rendering the cells competent. Likewise, the cells can be
repressed in RecBCD activity, for example, by repressing its
expression if using a repressible promoter, or by inducing the
expression of a RecBCD inhibitor, if driven by an inducible
promoter.
Method of Circularizing DNA
[0080] In another aspect, the present invention describes a method
of circularizing a linear DNA molecule comprising at least two loxP
sites. The method comprises introducing the linear DNA molecule
into an isolated host cell which transiently expresses the Cre
recombinase protein and which is at least transiently repressed for
RecBCD activity. The linear DNA molecule is circularized in the
cell by the Cre recombinase protein.
[0081] The linear DNA molecule is circularized by the site-specific
recombination at the two loxP sites. It will be appreciated by
those of skill in the art that, in addition to circularizing a
single linear DNA molecule, the method described herein can be used
to circularize a plurality of linear molecules, each containing at
least two loxP sites. Although expected to occur less frequently,
site-specific recombination and circularization of a plurality of
linear molecules can nevertheless be achieved using the methods
described herein, and such events can be identified using methods
known in the art (e.g., selection for circularization using
selectable markers present on each of the two linear
molecules).
[0082] In embodiments in which a plurality of DNA molecules is
joined and circularized, it is not necessary that all the DNA
molecules be introduced into the cell at once. For example, the
methods described herein can be used to join two molecules, one of
which was already present within the cell. Thus, it is only
necessary that one linear DNA molecule be introduced into the
cell.
[0083] The cells described herein transiently express or contain
the Cre recombinase protein. For the methods described herein, it
is only necessary that the cells express or contain the Cre
recombinase protein at the time that a linear DNA molecule is
introduced into the cell.
[0084] For the methods described herein, a higher degree of
circularization can be achieved by reducing exonuclease activity at
the time the linear DNA molecule is introduced into the cell.
Circularization methods described herein are enhanced by reducing
the exonuclease activities, for example of the RecBCD enzyme.
Therefore, in one embodiment, the cell lacks a functional recB
gene. In another embodiment, the cell lacks a functional recC gene.
In still another embodiment, the cell is transiently repressed for
RecBCD activity, in particular at the time that the linear DNA
molecule is introduced into the cell.
Kits
[0085] Also described herein are kits useful for the
circularization of linear DNA molecules. The kit comprises cells
described above, and optionally an instruction manual and packaging
material therefor. The cells can be provided as competent cells,
into which linear DNA molecules can be introduced.
[0086] The kit can also comprise primers, buffers, and additional
reagents which can be used to generate linear DNA molecules which
can be circularized according to the methods described herein.
[0087] The following are provided for exemplification purposes only
and are not intended to limit the scope of the invention described
in broad terms above. All references cited in this disclosure are
incorporated herein by reference.
EXAMPLES
Example 1
Inter-Molecular Ligation and Molecular Cloning Using Univalent
Topoisomerase-Bound DNA
[0088] An insert nucleic acid molecule, for example, a PCR product,
can be generated by PCR using a primer set consisting of a
5'-primer and 3'-primer. Two vector nucleic acid molecules, for
example, a left vector arm and a right vector arm are prepared such
that a topoisomerase enzyme (TOPO) is covalently bound only to one
end of a nucleic acid molecule to form a univalent topoisomerase
vector molecule. PCR primers for generating an insert molecule can
be synthesized to possess either a hydroxyl group or phosphate
group at each of the 5'-ends. A hydroxyl group permits ligation to
topoisomerase-bound DNA while a phosphate group prohibits such
ligation.
[0089] For non-directional ligation of a PCR insert molecule to,
for example two vector arms, both PCR primers will possess
5'-hydroxyl groups. The PCR insert can ligate with the vector arms
to form four different types of ligation products: 1) left vector
arm (LVA)-insert molecule (I)-left vector arm (LVA); 2) right
vector arm (RVA)-insert (I)-right vector arm (RVA); 3) LVA-I-RVA;
and 4) RVA-I-LVA. Only the LVA-I-RVA and RVA-I-LVA create viable
replication competent entities (FIG. 1).
[0090] For directional ligation of a PCR insert molecule to, for
example, left and right vector arms, one PCR primer possesses a
5'-hydroxyl group and the other PCR primer possesses a 5'-phosphate
group. The PCR generated insert molecule is generated and is first
ligated to one vector arm, for example, a LVA to create a
LVA-I-5'-phosphate molecule. The 5'-phosphate end of this molecule
is unable to ligate to the LVA or RVA because the vector arm sites
to which the TOPO is bound contain a 3'-phosphate. This molecule is
then dephosphorylated to create to LVA-I-5'-OH. The LVA-I-5'-OH
molecule is then ligated to the other vector arm (RVA) to form
LVA-I-RVA (FIG. 2). Once the ligated insert/vector molecule
described above has been constructed, the two vector arms can be
non-covalently or covalently joined to one another, at the ends
distal to the covalently attached topoisomerase polypeptide (i.e.,
their free ends), by a number of methods such that a circular
molecule is formed. Such methods include, for example, ligase
enzyme mediated ligation, complementary sequence annealing,
topoisomerase mediated ligation, in vitro or in vivo site-specific
recombination, or in vivo homologous recombination.
Example 2
Directional Molecular Cloning Using Topoisomerase and a Ligase
Enzyme
[0091] A nucleic acid insert is generated using, for example, a
pair of PCR primers wherein one primer (P1) has a hydroxyl group at
its 5'-end (OH-P1) and the other primer (P2) has a phosphate group
at its 5'-end (P2-P) (see FIG. 3). The insert molecule is generated
by PCR. A linear vector nucleic acid is prepared such that it has
TOPO bound at one end (univalent TOPO-bound nucleic acid molecule);
the other end of the linear vector nucleic acid comprises a
substrate for ligation (a 3'-OH) to be mediated by a ligase enzyme.
In a single incubation, the PCR insert can be ligated to the
TOPO-end of the linear vector nucleic acid via TOPO-mediated
ligation and to the other end of the linear vector nucleic acid via
a ligase enzyme-mediated reaction. The product of the ligation is
transformed into an appropriate host cell. A cloning event mediated
by both topoisomerase and DNA ligase is unidirectional. The
hydroxyl or phosphate group at the 5'-end of the PCR primers
determines the directionality of the insert.
[0092] A second approach involving a topoisomerase- and
ligase-mediated ligation comprises generation of an insert by for
example, PCR. Where PCR is used to generate an insert, a pair of
PCR primers where one has a hydroxyl group at its 5'-end (HO-P1)
and the other has a phosphate group at its 5'-end (P2-P) (see FIG.
4). A vector, such as two vector nucleic acid arms, can be prepared
such that one vector arm has a TOPO bound at one end (univalent
TOPO-bound DNA molecule) and the other vector arm has a substrate
for ligation at one end. In a single incubation, the PCR insert is
ligated to the one vector arm with a TOPO end via TOPO-mediated
ligation and to the other vector arm with the ligation-ready end
via ligase enzyme-mediated reaction. The product of the ligation is
transformed into an appropriate host cell. The cloning event
mediated by both topoisomerase and DNA ligase is unidirectional.
The hydroxyl or phosphate group at the 5'-end of the PCR primers
determines the directionality. The other ends of the two vector
arms are then joined by any of the methods described above. Using
this cloning method the ligation products comprised of RVA-I-RVA or
LVA-I-LVA should not be formed, but in the event that some do
occur, such ligation products are incapable of subsequent
replication and propagation.
Example 3
Molecular Cloning Using Topoisomerase and cos Ends
[0093] A method of molecular cloning using topoisomerase and cos
ends can comprise a vector, where such a vector may consist of two
vector arms, with each arm consisting of one TOPO-end and one cos
end. cos refers to the cohesive ends present at the termini of
bacteriophage lambda. An insert, such as a PCR insert, can be
generated using primers comprising 5'-OH termini. The PCR insert
can be ligated to a TOPO-end of the two vector arms by DNA
topoisomerase (see FIG. 5). Ligation events that result in
LVA-I-LVA or RVA-I-RVA cannot subsequently be propagated. The
product of the ligation can be transformed into a suitable host.
The distal ends of the vector arms contain terminal cos sites that
are readily annealed to one another in E. coli host cells by virtue
of their explicit sequence. cos sites do not anneal in vitro at
room temperature.
[0094] This method of cloning can be directional or
non-directional. In the case of non-directional cloning, an insert
comprises a 5'-hydroxyl ends and can be ligated to, for example,
two vector arms in a single reaction. For directional cloning, an
insert can be generated by, for example, PCR wherein one PCR primer
has a 5'-hydroxyl group and the other PCR primer has a 5'-phosphate
group. Thus, the resulting PCR insert will contain one 5'-hydroxyl
end and one 5'-phosphate end. The insert is to be ligated
sequentially, first to a left vector arm containing a TOPO bound
end followed by dephosphorylation of the 5'-phosphate of the insert
and then ligation to the right vector arm containing a TOPO bound
end (FIG. 6).
[0095] The ligation product of the insert to the vector is a linear
molecule in vitro with two cos sequences at its end. It is
transformed into a host, such as E. coli more efficiently than a
circular molecule.
Example 4
Molecular Cloning Using Topoisomerase and LIC Ends
[0096] A method of molecular cloning using topoisomerase and LIC
ends can comprise a vector, such as two vector arms, each
consisting of one TOPO-end and one LIC end. An insert, such as a
PCR insert, can be generated using primers comprising two 5'-OH
termini. The PCR insert can be ligated to a TOPO-end of the two
vector arms by DNA topoisomerase (see FIG. 7). Ligation events that
result in LVA-I-LVA or RVA-I-RVA cannot subsequently be propagated.
The distal ends of the vector arms contain terminal LIC sites that
are readily annealed to a plasmid comprising LIC compatible
ends.
[0097] This method of cloning can be directional or
non-directional. In the case of non-directional cloning, an insert
comprising 5'-hydroxyl ends and can be ligated to, for example, two
vector arms in a single reaction. For directional cloning, an
insert can be generated by, for example, PCR wherein one PCR primer
has a 5'-hydroxyl group and the other PCR primer has a 5'-phosphate
group. Thus, the resulting PCR insert will contain one 5'-hydroxyl
end and one 5'-phosphate end. The insert is to be ligated
sequentially, first to the left vector arm containing a TOPO bound
end and followed by dephosphorylation of the 5'-phosphate of the
insert and then ligation to the right vector arm containing a TOPO
bound end (FIG. 8).
Example 5
Molecular Cloning into Lambda Vector
[0098] The vector can comprise lambda DNA vector arms (termed left
lambda arm (LLA)) and right lambda arm (RLA)). An insert, such as a
PCR generated insert, can be ligated to the lambda vector arms in a
directional manner or non-directional manner. In the case of
non-directional cloning, a PCR insert can be generated using
5'-hydroxyl PCR primers. The insert can be ligated to two lambda
vector arms in a single reaction. Ligation events resulting in
LLA-I-LLA or RLA-I-RLA cannot subsequently be propagated. For
directional cloning, one PCR primer has a 5'-hydroxyl end and the
other PCR primer has a 5'-phosphate end. Thus, the PCR insert is
comprised of one 5'-hydroxyl end and one 5'-phosphate end. The
insert can be ligated sequentially to the two lambda vector arms
with a dephosphorylation step in between as depicted in FIG. 9. The
ligated lambda construct can be packaged in vitro and transfected
into host cells such as XLI-Blue E. coli. A circular plasmid DNA
containing the insert of interest can be rescued from the lambda
vector using, for example, ZAP technology (Stratagene).
Example 6
Molecular Cloning Into a Linear Plasmid DNA Molecule
[0099] A vector can comprise vector arms of a linear plasmid such
as N15. An insert, such as a PCR generated insert, can be ligated
to the plasmid vector arms in a directional manner or
non-directional manner. In the case of non-directional cloning, a
PCR insert can be generated using 5'-hydroxyl PCR primers. The
insert can be ligated to two plasmid vector arms in a single
reaction (FIG. 10). Ligation events resulting in LVA-I-LVA or
RVA-I-RVA cannot subsequently be propagated. For directional
cloning, one PCR primer has a 5'-hydroxyl end and the other PCR
primer has a 5'-phosphate end. Thus the PCR insert is comprised of
one 5'-hydroxyl end and one 5'-phosphate end. The insert can be
ligated sequentially to the two plasmid vector arms with a
dephosphorylation step in between as depicted in the FIG. 11. The
linear DNA can be transformed directly into E. Coli. Alternatively,
the ligated plasmid construct can be packaged in vitro and
transfected into host cells such as XLI-Blue E. coli. A DNA
containing the insert of interest can be rescued from the vector
using, for example, ZAP technology (Stratagene).
[0100] A vector can also comprise a linear plasmid vector
consisting of a covalently bound topoisomerase polypeptide at one
end and a ligation substrate site at the other end (see FIG. 4).
Incubation of the vector with an insert molecule comprising 5'-OH
group on one end and a 5'-phosphate group on the other end, under
conditions sufficient for topoisomerase-mediated ligation and
ligase enzyme-mediated ligation results in a ligated circular
plasmid comprising the insert molecule. The plasmid can be
transformed into a host cell.
Example 7
Molecular Cloning Using Topoisomerase and Site-Specific
Recombination
[0101] A vector can comprise vector arms that comprise one TOPO-end
and one loxP end. The loxp site can be recombined with a second
loxP site in the presence of a Cre site-specific recombination
protein. An insert, such as a PCR generated insert, can be ligated
to the TOPO-end of the two vector arms. Such cloning can be
directional or non-directional. In the case of non-directional
cloning, an insert, such as a PCR insert can be generated from PCR
primers each comprising 5'-hydroxyl ends. An insert comprising two
5'-OH ends can be ligated to two vector arms in a single reaction
(FIG. 12). For directional cloning, an insert can be generated by,
for example, PCR wherein one PCR primer comprises a 5'-hydroxyl end
and the other PCR primer comprises 5'-phosphate end resulting in an
insert that comprises one 5'-hydroxyl end and one 5'-phosphate end.
The insert can be ligated sequentially to two vector arms with a
dephosphorylation step in between as depicted in FIG. 13. The
ligation product comprises a loxP site at each end of a linear
molecule. The linear molecule can be recombined into a circular
recombinant plasmid in vitro, for example using purified Cre
recombinase or in vivo by, for example transformation into an E.
coli host expressing Cre recombinase and a plasmid that has loxP
sites.
Example 8
Molecular Cloning Using Topoisomerase and Homologous Recombination
in Vivo
[0102] In vivo homologous recombination can be exploited to
transfer a ligated insert/vector of interest into a circular
plasmid vector. Homologous sequences flank a ligated insert/vector
of interest and are substantially identical to sequences of a
plasmid cloning vector. A ligated insert/vector of interest is
recombined into a plasmid cloning vector of choice via homologous
recombination between the homologous sequences flanking the ligated
insert/vector and in the plasmid cloning vector. An insert can be
generated with homologous sequences attached to each end by, for
example, synthesizing PCR primers with homologous vector sequences,
of for example, 30, 75, 100, 150, 200, 250, 500, or 1000 base pairs
and using the PCR primers to generate a ligated insert/vector with
homologous vector sequences flanking the ligated insert/vector of
interest. A ligated insert/vector molecule with homologous
sequences at the ends can also be generated by preparing
topoisomerase-bound homologous sequence elements and employing a
TOPO cloning scheme as outlined in FIGS. 14 and 15 for generating
an insert with homologous sequence elements on each end. A PCR
amplified insert containing TOPO ligated arms can be transformed
into host cells containing a cloning vector wherein homologous
recombination can occur. For efficient in vivo homologous
recombination, a recA+host strain can be used. To protect a linear
insert from degradation by endogenous exonuclease activities, the
ends of the insert can be modified to either inhibit or prohibit
exonuclease digestion events.
[0103] To achieve site-specific in vivo recombination, lambda
attachment sites can be employed in place of the homologous
sequences described above. In this scenario, lambda attachment
sites flank a ligated insert/vector of interest, which is generated
according to the PCR and TOPO cloning schemes described above. The
ligated insert/vector with the flanking lambda attachment sites is
transformed into host cells containing a cloning vector with lambda
attachment sites. Inside the host cell, the ligated insert/vector
then can be site-specifically recombined into a plasmid cloning
vector between the lambda attachment sites flanking the ligated
insert/vector and those sites in the plasmid cloning vector.
Example 9
Generation of Strains (Methods)
A. Preparation of Recombination Cassette by PCR
[0104] 120/122 targeting fragment: this PCR fragment has a cre
recombinase gene with a Chloramphenicol resistance (CAM) marker
flanked by FRT sites. There is a 78 bp 5' homology armn to the AraB
promoter with 1 nucleotide deleted, which results in a better
ribosomal binding site and a 89 bp fragment homologous to the araD
gene. The cassette was assembled with the EasyA enzyme blend
(Stratagene #600404) using outer primers BB110 and 113 and 10 fold
diluted inner primers BB 111 and 112 using pACYC-Cre and pKSF-Cam
as template. To add longer homology arms this 110/113 product was
reamplified using primers BB120 and 122.
B. Vectors with Recombination Cassettes
[0105] pUC18-98-128: insertion site of araBA-cre vector in from E.
coli strain BW#3 amplified by EasyA with BB128/098 and cloned into
PvuII cut T-tailed pUC18.
[0106] pUC18 ara-cre V3.0: Changed RBS in pUC18-98-128 and mutated
a putative promoter at the ara/cre junction, inverted Cam cassette.
2 PCR fragments were generated: 1. Using primers BB93 and 111 and
10 fold diluted bridging primer BB133 and 134 with sure cells and
pACYC-cre as template; PCR product was digested with MluI and EcoRI
2. Using primer BB 13 1 and 132 to amplify Cam cassette from
pKSF-Cam, cut with BglII and EcoRI. Both fragments were cloned into
Mlul/ BglII cut pUC18-98-128. A 3.7 kb AflII-BstXI fragment can be
isolated from this plasmid for targeting.
[0107] pUC18 ara-cre V3.0 GTG: Changed the ATG start codon of cre
with GTG by a QuikChange reaction using primers BB141GTG and
142GTG
[0108] pUC ara-gam-cre: cloned gam recBC inhibitor upstream of the
cre cassette by overlapping PCR assembly. Briefly, a cassette was
assembled using outer primers BB093 and BB111 with 10-fold diluted
inner primers BB215,216, 217, and 218 (FIG. 19) using XL-1 cells,
pKD46 and pACYC-cre plasmids as template. This PCR fragment was cut
with MluI and EcoRI and used to exchange the corresponding fragment
in pUC18 ara-cre V3.0. The RBS for the gam cassette was optimized
based on the deletion of a T at position -12 in the ara promoter to
generate as consensus RBS. In addition, GTG was kept as a start
codon for Cre and the start codon also is incorporated into a PmlI
site for future cloning purposes. An MluI/BstXI fragment was used
for recombination.
C. Bacterial Strains
[0109] Cre Expressing E. coli TABLE-US-00002 verfication of correct
integration size of PCR primer wt PCR Bacterial Strain Pedigree
genotype primer pair product pair product Sure:cre Sure carries
pACYC-cre, constitutive cre recombinase expression BW ara/cre#3
BW25113 cre driven by araB promoter with 117/118 507 117/121 686
good RBS Sure ara/cre Sure P1 transfer of ara locus from BW 117/118
507 117/121 686 #1, 2 and 3 ara/cre#3 BW ara-cre clones BW25113 cre
driven by araB promoter with 140/118 1434 117/140 1613 2.1, 2.2 and
12.2 weak RBS Sure V3.0 GTG clone 5 Sure Cre driven by ara
promoter, weak 140/118 1434 117/140 1613 RBS and GTG start Sure
3.2.5.8 Sure V3.0 GTG Cre driven by ara promoter, weak 140/118 1127
nd nd clone 5 RBS and GTG start, Cam sensitive sure 3.2.5.66 Sure
V3.0 GTG Cre driven by ara promoter, weak 140/118 1127 nd nd clone
5 RBS and GTG start, Cam sensitive DH10B:cre clone 8.1 DH10B P1
transfer of ara locus from Sure 140/118 1127 nd nd V3.0 GTG clone 5
DH10B:cre 812 DH10B:cre clone Cam selection marker and all 140/118
1127 nd nd 8.1 plasmids cured DH10B:ara-gam-cre DH10B:cre 812 Ara B
promoter driven gam and cre 117/118 900 nd nd with GTG on one
operon DH10B:ara-gam-cre DH10B:ara-gam- Ara B promoter driven gam
and cre 117/118 900 nd nd 20 StrataClone cre with GTG on one
operon, Cam- Cells sensitive Colonies from the recombineering
events were screened by PCR to verify the correct integration.
Primer BB118 primes in the cre gene, wheras the other primer (117
or 140 primes upstream of the homology region used. For some case a
third primer was used that would give a product from the wt gene
before recombination. This PCR fragment will disappear upon correct
integration
D. P1 Transduction
[0110] P1 lysates were prepared by diluting 100 .mu.l of a
bacterial ON culture into 10 ml LB/5 mM CaCl.sub.2/0.2% glucose.
After 1 hour shaking at 37.degree. C. 200 .mu.l of a P1 phage
lysate was added. Typically, cells lyse after 2-3 more hours. A few
drops of Chloroform were added and vortexed to complete lysis and
the supernatant was stored at 4.degree. C.
[0111] For transduction into a recipient strain 2 ml of a fresh ON
culture were spun down and resuspended in 2 ml MC Buffer (0.1M
MgSO4, 0.005M CaCl.sub.2). 400 .mu.l of cells in MC buffer were
incubated for 15 minutes at 37.degree. C. with 400 .mu.l of
different dilution of P1 lysate (typically 1:1, 1:5, and 1:50
dilutions). Cells were spun down and resuspended in 800 .mu.l
citrate buffer (0.1M, pH 5.5: 9.6 g citric acid, 4.4 g NaOH,
dH.sub.2O to 500 ml; adjust pH to 5.5 with ION NaOH, autoclave).
100 .mu.l were spread on Cam selective plates and incubated ON at
37.degree. C.
E. Recombination and Subsequent Removal of Markers and Plasmids
Recombination and Subsequent Removal of Markers and Plasmids
[0112] Competent cells were prepared for recombination by
transfection of pKD46 into BW25223 and DH10B-derived strains or
pKD46I into Sure or Sure-derived strains. It was noted that the red
genes in pKD46 can be induced with L-Arabinose despite those cells
being ara+. An ON culture in NZY/Amp was diluted 1:100 and grown to
OD 600 of 0.6 in NZY. pKD46 strains were induced with 10 mM L-Ara
for 15 minutes and pKD46I strains with 1 mM IPTG for 15 minutes.
Cells were spun down and was 5 times with 20 cell volumes water and
taken up in 1/100 of the original culture volume in 10% glycerol in
water. Cells can be frozen at -80.degree. C or 40 .mu.l are used
fresh for Electroporation with 50 to 100 ng of PCR targeting
product or gelisolated plasmid insert. Cuvettes with a 1 mm gap
were used at 1700 V, 25 .mu.F, and 200 Ohms. Cells were recovered
in 1 ml NZY for 1 hour at 37.degree. C. before plating 10 to 200
.mu.l on CAM selective plates.
[0113] The Cam selection marker was cleared by transformation of
the strain with a Flp recombinase expression plasmid pACYC-Flp-Gent
and selection with 15 ug/ml Gentamycin at 30.degree. C. Pooled
colonies were replated and individual colonies were screened for
loss of Cam -resistance. The pACYC-Flp-Gent plasmid was determined
very stable in all strains, but could be bounced out by
transformation with plasmid pLys upon selection for Cam-resistance.
The pLys plasmid is lost spontaneously if not selected for the
Cam-resistance marker. Again, individual colonies were patched to
screen for loss of Cam (pLysS), Gentamycin (pACYC-Flp-Gent), and
Ampicillin (pKD46 or 46I). Colonies sensitive to all 3 marker
needed to be screened for the absence of extrachromosomal DNA by a
miniprep extraction (Note: One clone, Sure 3.2.5.8, was found to
contain extrachromosomal DNA, although it was sensitive to all
three antibiotics.)
F. Preparation of Chemically Competent cells
Sure Strains:
[0114] 100 ml NZY were inoculated with 1 ml fresh overnight (ON)
culture and grown to an optical density (O.D.600) of .about.0.25 in
the shaker at 37.degree. C. For induction of the Cre recombinase, 1
ml of 1 M L-Arabinose was added and incubation continued for about
20 minutes, at which point the OD600 reached .about.0.35. The broth
was cooled on ice and pelleted at 3500 rpm at 4.degree. C. The
supernatant was discarded and the cell pellet was resuspended in 20
ml ice-cold FSB/Glycerol and kept on ice for 20 minutes. Cells were
spun again and the pellet was resuspended in 4 ml FSB/10%
glycerol/7%DMSO.
DH10B Strains:
[0115] 100 ml LB/20 mM MgSO.sub.4 were inoculated with 1 ml fresh
ON culture, Cells were grown at 37.degree. C. to OD600 of about
0.4. For induction of cre and or the recBC inhibitor, 1 ml of 1M
L-Arabinose was added and incubation continued for 20 minutes.
Broth was cooled on ice, pelleted at 3500 rpm at 4.degree. C. Cell
pellet was resuspended in 20 ml ice-cold PG buffer (30 mM
KC.sub.2H.sub.3O.sub.2 pH 5.8, 10 mM CaCl.sub.2, 50 mM MnCl.sub.2,
100 mM RbCl and 15% glycerol) and kept on ice for 5 minutes. Cells
were spun again and the pellet was resuspended 2 ml ice-cold MG
buffer (10 mM MOPS pH 6.5, 75 mM CaCl.sub.2, 10 mM RbCl and 15%
glycerol). Cells are aliquoted and kept on ice for a total of 60
min and frozen at -80.degree. C.
Example 10
Construction of the L-Arabinose-Inducible Cre Strain.
[0116] The arabinose operon was chosen for the controlled
expression of cre-recombinase. Expression of the B, A, and D genes
of the ara operon is tightly controlled by the product of the araC
gene, and induction is strictly dependent on the presence of
arabinose (Lee et al, 1987). In order to generate an integrated
ara-cre expression cassette, the araBAD genes were replaced with
cre recombinase by recombination. This technology utilizes the
.lamda. red gam recombination system to target chromosomal
integration by homologous recombination. The integration occurs
precisely at a site predetermined by the flanking sequences of the
integration insert. A 30 base pair region of homology is sufficient
to target integration (Datsenko and Wanner, 2000). Integration
events are typically selected for by a selection marker present on
the inserted fragment, which can subsequently be removed if it is
flanked by appropriate sites permitting site specific
recombination.
[0117] The overall strategy used for assembly of a PCR cassette for
recombination is described in FIG. 15. The cre recombinase was
inserted in place of the araA and araB genes. In addition, the
ribosomal binding site (RBS) was modified by deletion of one T 12
bp upstream of the ATG start codon. This results in a consensus
ribosome binding site (AGGAG) 7 bp upstream of the translational
start site. A chloramphenicol selection marker (Cam) was placed
downstream of the cre gene to be able to select for recombination
events. The Cam.sup.R gene was flanked by FRT sites to be able to
remove the selection marker by consecutive Flp recombinase
expression.
[0118] This expression cassette was introduced into the
recombination strain BW25113 expressing the .lamda.-red gam genes
by electroporation. The resulting Cam-resistant colonies
represented the desired integration event as confirmed by PCR using
primer annealing within the recombination cassette and outside of
the targeted homology region. The resulting araBAD::cre cam.sup.R
locus was transferred to Sure by P1 transduction. However, although
the resulting strains (Sure:ara-cre#1 and #3) clearly expressed
cre-recombinase upon arabinose induction, the cre expression level
in the absence of induction were still sufficient to interfere with
the purification of LoxP containing plasmids (FIG. 16).
[0119] In order to reduce the uninduced cre-expression level two
changes were introduced into the promoter fragment. First, the
ribosome binding site was reverted from the consensus sequence to
the original araB ribosome binding site. In addition, a putative
promoter sequence present in cre-recombinase ORF was removed that
might give rise to a possibly active cre-derivative lacking the
N-terminal 27 amino acids. Changes in the araB promoter cre
junction are shown below, with sequence changes highlighted. The
ribosome binding site is boxed and the start codon is underlined:
TABLE-US-00003 ##STR1##
[0120] A targeting cassette containing these modifications was
integrated in the genome of Sure by performing recombination
directly in this strain. Although the derived strains expressed
arabinose inducible cre recombinase at apparently lower levels,
purification of loxP containing plasmids from this strain was still
impaired (data not shown). We therefore attempted to further reduce
uninduced cre-expression by changing the translational start codon
form AUG to GUG. Replacement of AUG with GUG typically reduces
expression level in E. coli about 10-fold.
[0121] The recombination cassette containing these additional
modifications was integrated into the genome of Sure by direct
recombination. All chloramphenicol resistant colonies derived
contained the desired integration event as confirmed by PCR. One
clone that had spontaneously lost the .lamda.-red/gam expression
plasmid pKD46I (Sure 3.2.5 ) was characterized further.
[0122] When tested for Cre-expression with the tester plasmid
pKSL-cam, no Cre-activity could be detected in uninduced Sure 3.2.5
(FIG. 18) although the strain is proficient for cre activity upon
induction (not shown). The tester plasmid pKSL-cam contains the
chloramphenicol resistance marker flanked by two LoxP sites.
Cre-mediated recombination results in the loss of the loxP-flanked
segment. Consistent with this result, LoxP containing plasmids
(pKSL-cam and pBA3.3) could be isolated with yields comparable to
the control plasmid pUC18.
[0123] The chloramphenicol resistance marker of Sure 3.2.5 was
removed by FLP mediated recombination, resulting in two independent
isolates (Sure 3.2.5.8 and 3.2.5.66.).
Example 11
Stability of Lox-Containing Plasmids in the Cre-Inducible Sure
3.2.5 Strain
[0124] Maintenance of loxP site containing plasmids in a
cre-expressing host is known to induce deletions involving the loxP
site. In order to evaluate the stability of a loxP containing
plasmid in Sure cells, two .beta.-galactosidase expressing plasmids
(PCH110 and pCAD-.beta.gal-BT) were introduced into the following
strains: [0125] Sure-1 (Stratagene), [0126] Sure 3.2.5.66 where cre
had been induced for 30 min before the cells were made competent,
and [0127] the Sure:cre strain that constitutively expresses cre
from the pACYC-cre plasmid.
[0128] The tester plasmids contain the full length
.beta.-galactosidase open reading frame (3.7 kb) with either no
LoxP sites (pCH110) or a loxP immediately 5' to the ORF
(pCAD-.beta.gal-BT). Deletions involving the .beta.-gal ORF result
in white colonies when the host cells are plated on x-gal plates.
The control vector CH110 (no loxP site) did not give rise to white
colonies in any of the host cells. In contrast, pCAD-.beta.gal-BT
transformed into the Sure:cre strain resulted in the appearance of
white colonies in with an average frequency of 6.7% (Table 1). No
white colonies were observed with this vector in conventional Sure
cells, demonstrating the cre-dependence of the deletion events.
Using the same tester construct only one white colony was observed
in one experiment in strain Sure 3.2.5.66, corresponding to a
24-fold improvement of stability compared to Sure:cre.
TABLE-US-00004 TABLE 1 Stability of loxP containing plasmids is
improved in Sure 3.2.5.66 CH110 CAD-.beta. gal-BT sample strain
white blue white blue Exp. 1 sure:cre 0 300 14 200 sure 0 300 0 163
sure 3.2.5.66 0 400 1 150 Exp. 2 sure:cre 0 260 8 128 sure 0 200 0
200 sure 3.2.5.66 0 170 0 200
Table 6: Plasmids CH110 and CAD-.beta.Gal-BT were diluted and
transformed into Sure, sure:cre and Sure 3.2.5.66. Transformation
was plated on Amp with X-Gal and IPTG. Plates were analyzed for
wild type blue and mutant white colonies after ON at 37.degree.
C.
Example 12
DH10B-Derived Strain for Circularizing the Linear Topoisomerase
Vectors
[0129] Nuclease activity by a functional RecBCD enzyme is expected
to reduce the overall circularization efficiency as the nuclease
would destroy the linear DNA molecule prior to circularization.
However, RecBCD positive cells may have additional advantages,
including generally faster dividing times, and ability to
incorporate a recA mutation without compromising cell stability.
Therefore, the following experiments were performed in order to
determine whether a RecBCD positive cell.
[0130] First, the ara::cre cassette was transferred from Sure
3.2.5.66 cells by P1 transduction into DH10B This strain is F- and
recA, readily produces blue and white colonies by
.alpha.-complementation. It can be made competent very efficiently
and forms large colonies after 16 hours at 37.degree. C. After P1
transduction, the cam marker was removed by Flp mediated
recombination, resulting in the strain DH10B-812. This strain was
made chemically competent using the TFB method after induction of
Cre expression with arabinose and compared to Sure 3.2.5.8 cells in
the efficiency of cloning the 1.8 kb Cam-OFP test insert in pBA 3.3
Topoisomerase-charged vector. As shown in Table 2, DHIOB-812 was
comparable to Sure 3.2.5.8 as a recipient strain for Topo cloning.
This is likely due to the superior transformation efficiency of
DH10B-812, which was almost 10-fold higher than the efficiency of
Sure 3.2.5.8. When normalized for transformation efficiency, Sure
3.2.5.8 was about 6-fold more effective than DH10B-812 establishing
colonies from linear Topo ligation product. This is consistent with
the lack of exonuclease activity in the Sure strains.
TABLE-US-00005 TABLE 7 Normalized cloning efficiency for the
DH10B:cre is lower than Sure 3.2.5.8 50 ul average ratio 5 ul
Amp/X- Transformation OFP over pUC Amp/X-gal/IPTG gal/IPTG 15 ul
pUC 19 efficiency per 300 ul host OFP white blue blue (0.5 pg) per
1 ug transformation DH10B:cre 812 81 0 0 6 226 4.21E+08 1.25 95 2 0
5 195 Sure 3.2.5.8 61 2 0 3 34 4.60E+07 7.50 54 1 0 2 12
Table2: 10 ng of 1.8 kb Cam-OFP test insert was ligated to 1 ul of
pBA 3.3 and 2 .mu.l of the ligation reaction (33%) was used
to-transform the indicated strains. The indicated volume (out of
300 .mu.l) was plated on Ampicillin plates supplemented with X-Gal
and IPTG. 10 pg of pUC19 served as transformation control. The
rightmost column displays the number of insert-containing (OFP+)
colonies per number of pUC transformants.
Example 13
DH10 Strain for Circularizing the Linear Topo Vector with a recBC
Inhibitor
[0131] Based on the results above, it can be projected that high
efficiency cloning strains like DH10B containing a recBC mutation
would be more effective host strains for linear topo-ligation
products. However, E. coli with mutations in recABC are very
unstable, presumably because DNA breaks cannot be repaired. An
alternative approach was attempted in which transient repression
RecBCD activity was achieved, generated by transiently expressing
inhibitors of recBC in a recA host. The recBC inhibitor from the
T3-phage (gene 5.9) and the .lamda.-gam gene were introduced into
the ara-cre expression cassette, in effect generating an arabinose
inducible cre/recBC inhibitor operon. In the first attempt, the
recBC inhibitors were inserted 3' to the cre recombinase. However,
these strains did not result in improved circularization efficiency
(not shown). In contrast, in case of the .lamda.-gam gene,
reversing the order of recBC inhibitor and cre recombinase
(DH10B:ara-gam-cre20; F- mcrA .DELTA.(mrr-hsdRMS-mcrBC)
.phi.80dlacZ.DELTA.M15 .DELTA.lacX74 deoR recA1 endA1
.DELTA.araBAD:: .lamda.gam-Plcre ga[U galK rpsL nupG.lamda.k- tonA)
resulted in a dramatic improvement in circularization efficiency
(not shown). The resulting strain, DH10B:ara-gam-cre, was as
efficient as Sure 3.2.5.8 with regard to the efficiency to
establish a colony from a linear Topo ligation product.
Sequence CWU 1
1
4 1 6 DNA Vaccinia virus 1 ctcctt 6 2 34 DNA Artificial sequence
LoxP site consensus sequence 2 ataacttcgt ataatgtatg ctatacgaag
ttat 34 3 53 DNA Artificial sequence AraB promoter Cre junction 3
ggaggaggaa catatgtcca atttactgac cgtacaccaa aatttgcctg cat 53 4 54
DNA Artificial sequence Modified araB promoter Cre junction 4
ggatggagga aacgatgtcc aacctgctga ccgtacacca aaatttgcct gcat 54
* * * * *